Flock house virus B2 protein Suppression of RNA Silencing



3D structure of insect Flock House virus protein B2 complexed to siRNA

Background
RNA silencing is a gene inactivation system in many eukaryotes that relies on tiny RNAs as the targeting molecules. One function of RNA silencing, which is also called post-transcriptional gene silencing (PTGS) or RNA interference (RNAi), is to act in surveillance against molecular parasites, such as viruses. Double-stranded RNA triggers the RNA silencing pathway and many viruses use a double-stranded RNA to replicate their genome. Various viruses have developed evasion techniques to circumvent this surveillance system. In one such evasion strategy, Flock House virus protein B2 suppresses the insect's anti-viral RNA-induced silencing response. siRNAs are generally characterized by their short length (21–26 nt), 2 nt, 3′ overhanging ends, and 5′ phosphate groups. The most efficient silencing is obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3' overhang (see the Tuschl lab's guide for designing siRNAs).

A structural study has revealed how Flock House virus protein B2 directly binds double-stranded RNAs that are the size of siRNAs and longer.

Results
The X-ray crystal structure of Flock House virus protein B2 complexed with double-stranded RNA includes both and  in the complex.

The RNA in the complex is double-stranded and in A-form. The synthetic R N A substrate mimics a small double-stranded siRNAs that occur in the double-strand RNA-induced RNAi silencing pathway. The duplex region here is 18 nts and a biological siRNA has typically a 19 nt duplex and also has additional 2-nt 3' overhangs. Here is a schematic illustration of the two strands of the double-stranded RNA in this structure:

5'-pGCAUGGACGCGUCCAUGC-OH-3' ||||||||||||||||||      3'-OH-CGUACCUGCGCAGGUACGp-5'

Flock House virus protein B2 (FHV B2) binds the double-stranded RNA as a dimer. Individual monomers of the B2 dimer are colored tan and purple.

Each monomer of FHV B2 is made of three alpha-helices, two long ones and and short one perpendicular to the long ones.

The FHV B2 dimer forms a four-helix bundle. An anti-parallel association of the two B2 monomers forms this four-helix bundle using two helices from each monomer. The third helix in each monomer caps the ends of the bundle. [ Note: this view generates a substantial surface which may take half a minute to calculate. ]

The four-helix bundle of the FHV B2 dimer has a unique proline acting to stabilize it. Unlike canonical left-handed antiparallel four-helix bundles (such as 1ax8) which have a interhelical angle of about 20°, the monomers of B2 are almost linear relative each other. A proline (P12)  kinks helix 1 of the monomer and the unusual feature stabilizes the non-canonical four-helix bundle. Underscoring the unusual nature of this proline is that it is found in a position highly unfavorable based on typical four-helix bundles and that it causes programs for predicting secondary structure to fail to predict the observed helix 1.

B2 contacts the phosphate backbone and ribose sugars to bind the RNA in a sequence independent manner along the whole length of the protein span. Although there is some interaction with the deep and narrow major groove, the majority of the interaction by B2 with RNA is with the shallow and wide minor groove. .
 * B2 makes major groove contacts using two basic residues (K47 and R54)  to contact the phosphate backbone.
 * Continuing out from the major groove contacts, B2 makes minor groove contacts using <font color = 'aqua'>residues 40, 44, 55, and 58 .
 * Finally, the contacts bridge the minor groove and B2 makes contacts in the minor groove on the opposite strand using <font color = 'red'>residues 33, 36, 37, and 62 , which interact with both the phosphates and 2'-hydroxyl.

Conclusions
FHV B2 functions as a dimer to bind to one face of double-stranded RNA in a sequence-independent manner.

Though the four-helix bundle is a common motif (see 1ax8,1a7m,1c03,1urq, 2vrz and other four-helix bundle structures, for examples), recognition of the double-stranded RNA by the B2 four-helix bundle revealed novel mode of RNA binding. Most likely, the B2 protein of Nodamura virus adopts a similar structure as almost all the RNA-contacting residues and Proline 12 are conserved between the two proteins despite marginal sequence similarity.

The solved structure shows how B2 could directly bind to siRNAs and block use of these intermediates in a defense response to viral attack. Additionally, the structure data and biochemical characterization of B2 binding is consistent with double-stranded RNA longer than 18 bps binding multiple B2 dimers. Such a protective coating of vial double-stranded RNA would inhibit processing by Dicer in the host cell.

In conclusion, the biochemical and structural characterization suggests B2 could act at two stages of RNA silencing. B2 can inhibit both cleavage of dsRNA by Dicer as well as siRNA incorporation into the RNA-induced silencing complex and thus aid viral infection in two ways.

About this Structure
2AZ0 is a Protein complex structure of sequences from Flock House Virus. Full crystallographic information is available from OCA. The molecular weight of FHV B2 protein chain designated 'a' seen in the solved structure is 7.7 kDa (70 residues visible). The molecular weight of the FHV B2 protein chain designated 'b' seen in the solved structure is 7.8 kDa (71 residues visible). Total size of the FHV B2-RNA complex seen in the structure is 28 kDa. Biological FHV B2 is 106 amino acids and recombinant FHV B2 used to generate the crystals was residues 1-73.

Reference for the Structure
Dual modes of RNA-silencing suppression by Flock House virus protein B2., Chao JA, Lee JH, Chapados BR, Debler EW, Schneemann A, Williamson JR, Nat Struct Mol Biol. 2005 Nov;12(11):952-7. PMID:16228003

Related Structures and Topics

 * Suppression of RNA Silencing by Viruses
 * Plant Viral Protein p19 Suppression of RNA Silencing
 * Tomato aspermy virus protein 2b Suppression of RNA Silencing
 * 2az0 Flock house virus B2 protein bound to double-stranded RNA (dsRNA)
 * 2b9z Flock house virus B2 protein solution structure
 * 2zi0 Tomato aspermy virus protein 2b bound to siRNA
 * 1rpu Carnation italian ringspot virus p19 bound to siRNA
 * 1r9f Tomato bushy stunt virus p19 bound to siRNA
 * RNA interference

Additional Literature and Resources

 * The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition., Lingel A, Simon B, Izaurralde E, Sattler M. EMBO Rep. 2005 Dec;6(12):1149-55. PMID:16270100
 * Tour of p19 bound to an siRNA by Wayne Decatur, in an exploration-friendly interface that is adapted from Eric Martz's FirstGlance in Jmol
 * Structural basis for RNA-silencing suppression by Tomato aspermy virus protein 2b., Chen HY, Yang J, Lin C, Yuan YA, EMBO Rep. 2008 Aug;9(8):754-60. Epub 2008 Jul 4.PMID:18600235
 * Recognition of small interfering RNA by a viral suppressor of RNA silencing., Ye K, Malinina L, Patel D J, Nature 2003 426(6968):874-878. Epub 2003 Dec 3. PMID:14661029
 * Sizing up small RNAs., Jabri E, Nature Structural & Molecular Biology 2004 11: 112. PMID:14749769
 * Crystal structure of p19--a universal suppressor of RNA silencing., Baulcombe DC, Molnar A, Trends Biochem Sci. 2004 29(6):279-281. PMID:15276178
 * Plant viral suppressors of RNA silencing., Roth BM, Pruss GJ, Vance VB, Virus Res. 2004 Jun 1;102(1):97-108. PMID:15068885
 * Novel modes of protein-RNA recognition in the RNAi pathway. Lingel A, Sattler M, Curr Opin Struct Biol. 2005. 15(1):107-115. PMID:15718141
 * Nature Reviews RNAi collection
 * The Tombusvirus-encoded P19: from irrelevance to elegance.,Scholthof HB, Nature Reviews Microbiology. 22006 May;4(5):405-11. PMID:16518419